Synthesis, stereochemistry determination, pharmacological studies and quantum chemical analyses of bisthiazolidinone derivative

Article abstract of DOI:10.1016/j.molstruc.2016.07.089

A new compound (3) bisthaizolidinone derivative was synthesized by Knoevenagel condensation reaction. The structure of synthesized compound was elucidated by different spectral techniques and X-ray diffraction studies. The stereochemistry of the compound

Full text of DOI:10.1016/j.molstruc.2016.07.089

Journal of Molecular Structure 1127 (2017) 99e113
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, stereochemistry determination, pharmacological studies
and quantum chemical analyses of bisthiazolidinone derivative
Md. Mushtaque a , Fernando Avecilla , Zubair Bin Hafeez , Meriyam Jahan a
,
*
b
c
,
1
, 1
,
e
,
1
c
,
**
d
Md. Shahzad Khan , M. Moshahid A. Rizvi
Anurag Srivastava , Anwesha Mallik , Saurabh Verma
School of Physical and Molecular Sciences, Department of Chemistry, Al-Falah University, Dhauj, Faridabad, Haryana, 121004, India
Departamento de Química Fundamental, Universidade da Coru n~ a, Campus da Zapateira s/n, A Coru n~ a, 15008, Spain
Department of Biosciences, Jamia Millia Islamia, New Delhi, 110025, India
Department of Physics, Jamia Millia Islamia, New Delhi, 110025, India
Advanced Material Research Group, ABV-Indian Institute of Information Technology and Management, Gwalior, 474015, India
National Institute of Pathology, Safdarjung Hospital Campus, New Delhi, 110029, India
, Mohd. Shahid Khan ,
e
f
f
a
b
c
d
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2016
Received in revised form
A new compound (3) bisthaizolidinone derivative was synthesized by Knoevenagel condensation reac-
tion. The structure of synthesized compound was elucidated by different spectral techniques and X-ray
diffraction studies. The stereochemistry of the compound (3) was determined by 1He H NOESY, He H
1
1
1
17 July 2016
NMR COSY and single crystal X-ray diffraction studies as (Z, Z)-configuration. The computational quan-
tum chemical studies of compound(3) like, IR, UV, NBO analysis were performed by DFT with Becke-3-
Lee-Yang-Parr (B3LYP) exchange-correlation functional in combination with 6e311þþG(d,p) basis sets.
Accepted 20 July 2016
Available online 22 July 2016
5
¡1
The DNA-binding of compound (3) exhibited a moderate binding constant (K
b
¼ 1 ꢀ 10 Lmol ) with
Keywords:
Cytotoxicity
Cell cycle perturbation
DNA binding
hypochromic shift. The molecular docking displayed good binding affinity ¡7.18 kcal/mol. The MTT assay
of compound (3) was screened against different cancerous cell lines, HepG2, Siha, Hela and MCF-7.
Studies against these cell lines depicted that the screened compound (3) showed potent inhibitory ac-
Molecular docking and DFT studies
tivity against HepG2 cell (IC50 ¼ 7.5
Hela (IC50 74.83 M) cell lines, and non-toxic effect against non-cancerous HEK-293 cells
IC50 ¼ 287.89 M) at the concentration range (0e300) M. Furthermore, cell cycle perturbation was
m
M) followed by MCF-7 (IC50 ¼ 52.0
m
M), Siha (IC50 ¼ 66.98
mM),
¼
m
(
m
m
performed on HepG2 & Siha cell lines and observed that cells were arrested in G2/M in HepG2, and G0/
G1 in Siha cell lines with respect to untreated control. Hence, compound (3) possesses potent anti-
cancerous activity against HepG2 cell line.
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
creating havoc [3] for example, Asian countries like China and India
along with Russia contribute more than half of the total cancer
cases [4]. The current data of India show that every year, around 0.7
million people are losing their lives with one million new diag-
nosed cancer patients, which are estimated to be roughly doubled
by the year 2035 [4]. Similarly, it is predicted that by 2030,
approximately 20 million new cancer cases will be diagnosed
worldwide and about 13 million cancer patients will die from this
disease [3,5]. Indeed, many treatment modalities and drugs are
available in the market to curb this terrifying disease like bio-
alkylating (chloramethine) [6], anti-metabolic agents (fluorouracil)
Cancer is one of the deadliest diseases in human race ever seen
1]. According to world health organization (WHO), it is second
[
most lethal disease causing deaths both in developing and devel-
oped countries [2]. All the developed, developing and under
developed countries have constantly been afraid of this disease
because it devastates both manpower as well as economy; thus
** Corresponding author.
*
Corresponding author.
E-mail addresses: mush.chem@gmail.com (Md. Mushtaque), rizvi_ma@yahoo.
[7], anti-cancer antibiotics (doxorubicin) [8].
The anti-cancer drug development with less or no side effect is
very important for chemotherapy of cancer. The necessity of such
com (M.M.A. Rizvi).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.molstruc.2016.07.089
0
022-2860/© 2016 Elsevier B.V. All rights reserved.

100
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
potent anti-cancer therapeutic agents has led to discovery of small
synthetic molecules which have anti-cancer activity with lesser
side effects. Thiazolidone molecular scaffold is of great importance
in modern medicinal chemistry, and is known to exhibit a diverse
range of pharmacological activities like anti-cancer [9e12], anti-
bacterial [13e15] Fig. 1(a&b), anti-fungal [16e18], anti-viral
methylene (HeC]C-) proton confirmed the formation of com-
13
pound (3). In C NMR, the appearance of significant peaks due to
C]O and C]C at 166.56 ppm and 148 ppm chemical shifts
confirmed the formation of compound (3). In mass spectrometry,
þ
þ
þ
the presence of [MþH] , [Mþ2H] and [Mþ3H] peaks at 567.18,
568.19 and 569.18 showed the formation of compound (3). Addi-
tionally, the structure of the compound (3) was confirmed by X-ray
single crystal structure.
[19e22], and anti-HIV [23,24].
Previous studies on this class of molecule report excellent anti-
amoebic activity with a high cytotoxicity [25]. In the view of ver-
satile pharmacological importance, we herein, report the synthesis,
characterization, DNA binding, molecular docking and DNA, cell
cycle perturbation, and cytotoxicity. Also, a DFT study was carried
out for evaluation of its biological potency of compound (3).
2
.2. Stereochemistry and conformational properties
The determination of E or Z-configuration is important for
compound (3) due to the presence of exocyclic C]N and C]C
bonds. The configuration of compound (3) was determined by
2
NMR, the strong interactions were observed along off-diagonal
peaks. The doublet due to methyl group at the range of
1
1
1
1
1
1
D- He H COSY-NMR and He H NOESY-NMR. In He H COSY-
2
. Results and discussion
2.1. Chemistry
1.623e1.051 (ppm) chemical shift interacted with multiplet of CH at
the range of 5.051e4.982 (ppm) chemical shift. But no off-diagonal
peaks are present in the region of the aliphatic protons attached to
the nitrogen of thiazolidinone ring and aromatic protons
substituted at exocyclic nitrogen which could show correlation
among them, indicating that the position of aromatic moiety
adopted Z-configuration. Furthermore, there are strong in-
teractions among the protons of exocyclic nitrogen substituted ar-
omatic ring at chemical shifts range 7.432e7.349 ppm and 5-
arylidene aromatic protons at chemical shift 6.990 ppm which
confirms Z-configuration of exocyclic 5-arylidene. A Z-configura-
tion of aromatic ring attached to exocyclic C]C bond of thiazoli-
dinone derivative was further confirmed by a methine proton
signal which resonated at a higher chemical shift 7.632 ppm as a
0
0
0
The presented compound (3) (2Z, 2 Z, 5Z, 5 Z)-5,5 -(1,4-
phenylenebis (methanylylidene)) bis(3-isopropyl-2-(phenylimino)
thiazolidin-4-one), was synthesized by reported method [26]. The
compound (3) was prepared in absolute ethanol using tereph-
thaladehyde and thiazolidinone by Knoevenagel condensation re-
action as shown in Scheme 1. The compound (3) is stable at room
temperature and well characterized by different spectroscopic
techniques; CHNS analysis, FT-IR, H NMR, C NMR and mass
spectroscopy. The stereochemistry of compound (3) was deter-
mined by He H COSY-NMR and He H NOESY-NMR. Furthermore,
stereochemistry was confirmed by X-ray single crystal structure.
The appearance and disappearance of the characteristic bands
confirmed the formation of the compounds. In FT-IR, the appear-
ance of the significant bands at 3265.14 cm and 1240.83 cm
due to eNH and C]S confirmed the formation of compound (1). In
H NMR, the chemical shifts appeared at 8.267 ppm and 5.783 ppm
due to presence of eNH protons, confirmed the formation of
compound (1). Additionally, the compound (1) was confirmed by
C NMR. The presence of significant peak at 178.94 ppm was
assigned to C]S, indicated the formation of compound (1). In case
of intermediate compound (2), in IR, the appearance of character-
istic bands at 1715.24 cm and 1627.65 cm due to eC]O and
eC]N confirmed the formation of compound (2). In H NMR, the
1
13
1
1
1
1
1
ꢁ
1
ꢁ1
singlet in H NMR spectrum. The downfield chemical shift of the
methine proton was due to deshielding effect of carbonyl group.
Studies on E-configuration suggest the upfield resonance at
1
chemical shift 6.6 ppm or less than
NOESY H NMR, the C]N imino and the C]C exocyclic double
d 6.6 (ppm) [25e28]. In 2D
1
13
bond exhibited strong NOE signals at chemical shifts
7.432e7.349 ppm and 6.990 ppm due to the interaction of protons
of two aromatic rings attached to imine (C]N) and 5-arylidene,
indicating Z-configuration. No NOE signals were observed for
exocyclic N-substituted aromatic protons and aliphatic (chain
attached to the N-of thiazolidinone) protons which suggest that the
molecule adopted Z-configuration. Additionally, a study of X-ray
single crystal structure confirmed (Z, Z)-configuration Fig. 2.
ꢁ
1
ꢁ1
1
presence of singlet at chemical shift 3.701 ppm due to the presence
of-SeCH eC]O proton showed the formation of compound (2).
2
The lead compound (3) was also confirmed by same spectroscopic
techniques along with mass spectrometry and X-ray single crystal
structure. In FT-IR, the presence of characteristic bands at
704 cm and 1629 cm due to C]O and C]C confirmed the
formation of compound(3). In H NMR, the presence of character-
istic peak resonated at high chemical shift at 7.631 ppm due to
2.2.1. Discussion of crystal structure
ꢁ1
ꢁ1
0
0
0
1
ORTEP diagram for the compound (3) (2Z,2 Z,5Z,5 Z)-5,5 -(1,4-
Phenylene bis(methanylylidene))bis(3-isopropyl-2-(phenylimino)
thiazolidin-4-one), was shown in Fig. 3. The asymmetric unit of 1
1
a
b
CH3
O
O
O
HO
O
S
N
O
N
S
H
H
N
N
N
N
H
H
S
N
O
N
S
O
O
O
O
H C
3
OH
Fig. 1. (aeb): 4-Thaizolidinone derivatives (1 a & b) showing anti-bacterial.

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
101
NCS
H
H
NH2
CH3
CH3
N
N
a
+
H C
CH3
3
S
(1)
b
O
S
O
N
N
N
c
+
2
CH3
CH3
N
H C
S
3
N
CH3
O
S
H C
H C
3
3
O
N
O
(2)
(3)
Scheme 1. (a) Toluene, Room temperature (b) Chloroacetic acid, Anhydrous sodium acetate, EtOH, reflux (c) Piperidine, EtOH Reflux.
contains only one molecule of compound (3). A Z-configuration of
the exocyclic C]C and C]N bonds is observed in the structure. The
phenyl ring [C15eC16eC17eC18eC19eC20] is not entirely flat with
respect to the thiazolidinone rings[S1eC11eC12eC13eN2, torsion
ꢂ
angle, 15.95(7) and for S2eC22eC23eC24eN4, torsion angle,
ꢂ
1
8.55(7) ] and bonded to imine groups which are rotated with
respect to the thiazolidinone rings[S1eC11eC12eC13eN2, torsion
ꢂ
angle
respect
to
C1eC2eC3eC4eC5eC6,68.15(3)
and
S2eC22eC23eN3, torsion angle respect to C28eC29eC30e-
ꢂ
C31eC32eC33, 60.34(4) ]. The values of the bond lengths and an-
gles are in agreement with those found in the similar derivatives
[29]. Bond lengths and angles are summarized in Table (S1) (sup-
plementary data). The crystal structure packing is mainly due to
van der waals forces. Weak intermolecular hydrogen bonds be-
tween CH bonds of the phenyl rings and the methylidene groups
with O atoms of thiazolidin-4-one groups are observed in the
crystal packing (Fig. 3) [30]. Weak intramolecular and intermolec-
ular hydrogen bonds between the methyls of the propyl groups, O
atoms of thiazolidin-4-one groups and N atoms of the imine groups
determine the configuration of the structure around C]C and C]N
bonds Table 1. Also, weak hydrogen bonds between CH groups and
Fig. 2. X-ray single crystal structure of compound (3).
with help of optimized geometry and is represented in Fig. 4. NBO
analysis illustrates role of intra and intermolecular bonding in-
teractions. It also provides suitable base for investigation of charge
transfer or hyper conjugative interactions in molecular system. The
energy difference between interacting orbitals is proportional to
stabilization of orbital interaction. Hence, the strongest interaction
of stabilization occur between dominant donor (i) and acceptor (j).
Quantitatively, interaction between bonding and anti-bonding or-
bitals is described in terms of NBO basis, and is expressed by means
of second order perturbation interaction energy, E(2) [35]. This
energy depicts an estimate of the off diagonal NBO Fock matrix
element. The stabilization energy is calculated by following math-
ematical expression:
p
clouds of the terminal phenyl rings appear in the crystal packing
(
see Fig. 3) [31].
2.3. Computational details
The ground state optimization of compound (3) has been carried
out using DFT with Becke-3-Lee-Yang-Parr (B3LYP) exchange-
correlation function [32,33] in combination with 6e31þþG(d,p)
basis sets using Gaussian03 package [34] without any symmetry
constraint. The NBO and Mulliken population analysis are also re-
ported for the local minima of a molecule. For electronic excited
state calculations, TD-DFT/B3LYP method has been employed with
the same basis set considered for ground state optimizations. The
simulated IR spectrum was analyzed by GAUSSVIEW and VEDA 4
program to assign vibrational frequencies with high degree of ac-
curacy. Molecular electrostatic potential of the molecules have also
been presented to view electrophilic and nucleophilic reactive sites.
Fði; jÞ2
2
E ¼
D
Ei; j ¼ qi
(1)
εi ꢁ εj
where qi is donor orbital occupancy, εi and εj are diagonal elements
orbital energy) and F(i, j) is off diagonal Fock matrix. The NBO
(
analysis has been carried out on entitled compound (3) using
Gaussian 03 package at the B3LYP/6-311G(d,p) level for illustrating
intramolecular charge transfer and delocalization of electron den-
sity within molecule. The significant interactions between donor
NBO orbitals and acceptor orbitals of compound (3) are given in
2.3.1. NBO analysis
The NBO analysis was carried out by 6e311þþG(d,p) basic set

102
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
of the highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO). The small interfrontier
orbital of a molecule is chemically more active and has low kinetic
stability [36e39]. The highest occupied molecular orbital (HOMO)
directly corresponds to ionization potential. However, the lowest
unoccupied molecular orbital corresponds to electron affinity
(electron gain enthalpy) of a molecule [41]. The HOMO and LUMO
orbitals have been shown in Fig. 5(a). These two orbitals are very
significant to understand the potency of a molecule by calculating
chemical potential, global hardness and electronegativity. The
ionization energy and electron affinity are expressed as I ¼ -EHOMO
and A ¼ -ELUMO, respectively. The chemical potential and hardness
are derived by mathematical expression;
m
¼ -(I þ A)/2 and
h
¼ (I-
A)/2. The compound (3) has EHOMO ¼ ꢁ5.7eV and ELUMO ¼ ¡2.58eV.
Subsequently the associated and
m
h
are ¡4.15ev and ¡1.6eV,
respectively. The stability is presented by negative chemical po-
tential. The hardness of the title molecule is 1.6eV and believed to
be soft or chemically active. The energy gap between HUMO and
LUMO of molecule is 3.142 eV as shown in Fig. 5(b).
2.3.3. Relation between theoretical and experimental UVevisible
Due to high accuracy and low computational cost, TD-DFT is
Fig. 3. Crystal packing in the compound (3). Blue dashes lines show some CeH/O and
CeH$$$ hydrogen bonds. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
widely used of for electronic spectra transitions. The experimental
and simulated UVeVisible absorption spectra of compound (3)
have been shown in Fig. 6(aeb). The vertical excitation energies,
oscillator strength (f) and transition wavelength along with as-
signments have been mentioned in Table 3. The experimental
wavelength was observed at 400 nm having absorbance of 3.02.
However, its theoretical counterpart was calculated at 449 nm
associated with oscillator strength of 1.42. This electronic transition
is majorly associated with HOMO to LUMO with 86.5% contribution.
As it is clear from Fig. 7, HOMO electron density is delocalized over
the whole molecule, which means HOMO/LUMO transition re-
sults in the electronic density to confine at the central benzene ring.
Canonical molecular orbitals output from the G03 software package
suggests that these frontier molecular orbitals (FMOs) mainly
p
Table 2. It was found that lone pair of electron present on sulphur
atom delocalized into anti-bonding molecular orbitals of
p*(N3eC30) and p*(C32 e C33) through hyper conjugation and
gives stabilization energy of 23 kcal/mol and 21 kcal/mol. However,
lone pair of electrons present on N3, N7 and N10 interacts with
anti-bonding orbital of s*(S1eC30), p*(O2eC31) and p*(N13eC49)
through hyper conjugation, causing stabilization energy 25 kcal/
mol, 58 kcal/mol and 47 kcal/mol respectively. The lone pair of
electron present on O2 interacts with anti-bonding orbital
s
*(N7eC31) through resonance and gives stablization energy
7 kcal/mol. The -bonding electron of (C32eC33) interacted
with the anti-bonding orbital of *(O2eC31) through hyper-
conjugation, causing stablization energy 20 kcal/mol, and
bonding orbital of (C16eC18) interacted with the anti-bonding
orbital of *(C4eC8) through resonance and caused stabilization
consist of p orbital, hence this transition is
extended conjugation). Transition observed at 256 nm is qualita-
tively in good agreement to the calculated value of 256 nm which is
p/p* in nature
2
p
p
(
p
p-
*
both
p/p* and n/s in nature. The contribution of electronic
p
excitations from HOMO and HOMO-3 to LUMOþ2, LUMOþ5 and
LUMOþ1 contribute this transition. The other two important
transitions which are calculated in between these two extremes are
p
energy 21 kcal/mol. Thus, whole molecule is stabilized by hyper-
conjugation and resonance.
3
81 nm and 293 nm. The former result to the significant electronic
charge shifting from thiazolidinones to the central benzene ring
and is * in nature, this transition involves electronic excitation
from HOMO-2 to LUMO orbital having a contribution of 92%.
However, later one has combinatorial nature of n/ * and
transitions. The experimental electronic transitions at 370 nm and
2.3.2. Frontier molecular orbitals
p/p
The biological potency of a molecule can be described in terms
p
p/p*
Table 1
2
2
80 nm correspond to these calculated transitions of 381 nm and
93 nm.
Weak intermolecular and intramolecular hydrogen bonds in the compound (3).
D-H/A
d(D-H)
d(H/A)
d(D/A)
<(DHA)
C(7)-H(7C)/O(1)
C(8)-H(8A)/O(1)
C(8)-H(8C)/O(2)a
C(9)eH(9)/N(1)
C(14)eH(14)/O(2)
0.98
0.98
0.98
0.965(14)
0.930(15)
0.940(14)
0.955(16)
0.98
2.52
2.56
2.72
2.343(14)
2.953(15)
2.991(14)
2.367(16)
2.53
3.0691(15)
3.1381(16)
3.6578(17)
2.8595(15)
3.7467(14)
3.7865(14)
2.8660(15)
3.5017(18)
3.0446(16)
3.1290(16)
115.1
117.6
159.6
112.8(10)
144.1(11)
143.3(11)
112.1(11)
172.7
2.3.4. IR analysis
The compound (3) consists of 70 atoms which consequence to
204 normal modes of fundamental vibrations. This molecule be-
b
longs to C1 symmetry. Up to the best of our knowledge none have
reported the quantum chemical study for molecular structures and
IR-spectra of compound (3). The observed and calculated IR-spectra
have been presented in Fig. 8 and comparison has been demon-
strated in Table 4. The rigorous analyses for fundamental modes of
vibrations with B3LYP have been shown in Table 4. In our study, we
have scaled the B3LYP calculation with 0.98 to correct the theo-
retical error.
C(21)eH(21)/O(1)c
C(25)eH(25)/N(4)
d
C(26)-H(26A)/O(1)
C(26)-H(26C)/O(2)
C(27)-H(27A)/O(2)
0.98
0.98
2.48
2.58
116.5
115.8
Symmetry transformations used to generate equivalent atoms.
a
ꢁ
xþ1,ꢁy,ꢁzþ1.
b
c
x,yþ1,z.
x,yꢁ1,z.
d
ꢁx,ꢁy,ꢁzþ1.
2.3.4.1. CeH vibrations. The CeH stretching vibration from

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
103
Fig. 4. XRD structure and optimized geometry of compound (3) Numbers display on atoms of optimized molecule is natural charges obtained by NBO calculation.
ꢁ
1
Table 2
dispersed on modes at 3223, 3212, 3207, 3201.20 and 3188 cm
The C56eH57 and C52eH53 stretching vibration contribute at
.
Second order perturbation theory analysis of Fock matrix in NBO basis corre-
sponding to some the intra-molecular bonds of compound(3).
ꢁ1
ꢁ1
wavenumber 3160 cm and 3126 cm with 74% and 90% contri-
butions. The C20eH21 stretching contributes at wavenumber
Donor(i)
Acceptor(J)
E2(Kcal/mol)
ꢁ1
3
055 cm with 86% contribution. C52eH53, C52eH54, C56eH58
LP(2)S1
LP (2)S1
LP(1)N3
LP(1)N7
LP(1)N10
LP(2)O2
p
p
*(N3 e C30)
*(C32eC33)
23
21
25
58
47
27
20
21
ꢁ
1
and C56eH59 stretching contribute wavenumber at 3126 cm
s*(S1eC30)
with 12% contribution. The C61eH62 stretching contributes at
212 cm with 72% contribution and rests have been given in
p*(O2 e C31)
ꢁ1
3
p*(N13 e C49)
Table 4. These vibrational modes in particularly assigned in our
s*(N7eC31)
ꢁ1
ꢁ1
p
p
(C32eC33)
(C16eC18)
p
p
*(O2eC31)
*(C4eC8)
experimental observation collectively at 3089 cm , 3034 cm ,
ꢁ1
ꢁ1
ꢁ1
2977 cm , 2936 cm and 2880 cm . The other mode of vibra-
tions like bending, torsion and out of plane of bending modes has
been summarized in Table 4.
different location of compound (3) falls in the region of
3
054e3201 cm 1. The CeH vibration of terminal methyl group
ꢁ
2
.3.4.2. C¼O vibrations. Theoretically, the OeC stretching vibration
ꢁ1
ꢁ1
contributes to the peaks at 3055 cm , 3123, 3126 cm
and
of C]O of thiazolidinone ring is assigned at wavenumber
ꢁ1
ꢁ
1
3160 cm . The CeH stretching vibrations of benzene rings are
1783 cm . However, its experimental counterpart was observed at
Fig. 5. Frontier molecular Orbitals of compound (3).

104
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
Fig. 6. (a&b): Experimental (a) and theoretical (TFDFT-B3LYP/6-31g(d,p)) UVeVis spectra of compound(3).
Table 3
DFT calculation have been compared with the experimental results.
From Tables TS1 & TS2 (Supporting information), it was observed
that the theoretical and experimental results are almost same. A
few deviations from experiment results have also been observed.
Comparative study between experimental and theoretical UV-Visible in chloroform
background of compound(3).
Experimental
TD-B3LYP
Oscillator Strength
1.4
ꢂ
The experimental angle of C (11)-N (1)-C (1) was 117.70 . However,
l
nm
Abs
l
nm
Assignments
ꢂ
its theoretical bond angle was 123.123 . The bond angle of C (24)-
400
3.02
449
381
293
HOMO e LUMO(86.5%)
ꢂ
N(4)-C (28) was observed at 118.31 . However, its theoretical bond
p/p*
ꢂ
angle was 123.123 . The bond angle of C (13)-C (14)-C (15) was
3
70
80
2.80
1.44
0.34
0.19
HOMO-2 e LUMO(92%)
nd
HOMO-6 e LUMO(54.2%)
n/
HOMO-1e LUMOþ1(31%)
ꢂ
ꢂ
p*
129.63 and its theoretical bond angle was 131.758 .
2
p
*
2.3.6. Molecular electrostatic potential (MEP)
Molecular electrostatic potential (MEP) is a useful descriptor to
p/p*
HOMO-3e LUMOþ1(21.4%)
describe the chemistry of a designed molecule in quantum chem-
ical simulation and is based on electron density (ED). It helps in
finding the active electrophilic and nucleophilic sites along with
hydrogen bonding [40]. The mapping of electrostatic potential to
the total electron density in the framework of ab-initio calculation
results the 3D image visualization in GaussView4 graphical inter-
face. The electron rich (negative potential) region has been coded
with red color and electron deficient (positive potential) region has
been coded with blue color. The zero potential surfaces have been
coded with Green color. Other colors have represented in between
the limits of red and blue. The electrophilic potential increases in
the order of red < orange < yellow < green < blue. It could be
inferred from Fig. 9 that, dangling oxygen at 4-thiazolidinone-rings
is an electron rich center and the terminal hydrogen at benzene
ring has a positive electron cloud and could be consider as pro-
tonation of these hydrogen atoms. So these sites can serve as a good
attractor for electrophilic and nucleophilic species and are
comparatively more chemically active than other molecular
regions.
p/p*
2
56
2.04
256
0.14
HOMO e LUMOþ2(22.2%)
p/p*
HOMO e LUMOþ5(19.6%)
*
n/s
704 cmꢁ1. The out of plane vibration of O2eC32eN7eC31,
O2eC32eN7eC31, O6eC47eN10eC48, O2eC32eN7eC31 of thia-
1
ꢁ1
ꢁ1
zolidinone ring contribute wavenumber at 735 cm and 725 cm
ꢁ
1
and 752 cm respectively. The experimental results for these vi-
ꢁ1
ꢁ1
brations were observed at 735 cm and 764 cm respectively.
2
.3.4.3. C¼C vibrations. The theoretical stretching vibrations asso-
ꢁ1
ciated with alkenic carbon (C]C) was found at 1630 cm with
contribution of 50%. However, experimental vibration was
ꢁ
1
observed at 1629 cm
.
2
.3.4.4. CeNeC vibrations. The CeNeC bending vibration due to
C4eN3eC30 and C28eN7eC31 contribute wave number at
3. Pharmacological activities
ꢁ1
ꢁ1.
9
05 cm . The experimental result was found at 911 cm
3
.1. DNA binding
2
.3.4.5. C¼N vibrations. The calculated CeN stretching vibrations
are observed in combination with other vibration modes and are
dispersed in the range of 1136e1377 cm . The CeN stretching
vibration due to N3eC30, N7eC30 and N7eC31 contribute at wave
number 1315 cm . The experimental wave number was observed
at 1592 cm . The other modes of vibrations have been depicted in
Table 4.
DNA is one of the most important targets for many pharma-
ꢁ1
ceutically active agents including anticancer drugs. Many organo-
metallic and organic moieties are well known which bind to DNA in
different ways [41e43]. Hence, study of interaction of new com-
pounds with DNA would be a promising step for the development
of new drugs. For this purpose, UVeVisible spectroscopy is one of
the most commonly employed techniques [43]. In UVeVisible
spectrophotometric titration, hypochromic and hyperchromic
shifts with change in wavelength, absence or presence of isosbestic
points are the characteristic of interactions of compounds with
ꢁ
1
ꢁ1
2
.3.5. Comparison between theoretical and experimental bond
angles and bond length
The theoretical bond angles and the bond lengths obtained by

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
105
Fig. 7. The frontier molecular orbital in terms of HOMO and LUMO obtained at contour value of 0.02.
DNA [44,45]. Classically, it has been well accepted that hypo-
chromism and bathochromism are the indications of non-covalent
binding, however, hyperchromism is the indication of covalent
binding [38,45]. As seen in Fig. 10(a & b), upon interaction of
compound with Ct-DNA, the peaks in the range of 200e450 nm
appeared, which underwent bathochromic shift with hypochrom-
ism, and this indicated the formation of compound-DNA adducts,
mainly through non-covalent manner [43]. Present study showed
phase distribution Fig. 11(aed), 28%, 33% and 35% respectively ar-
rest in G2/M as compared to untreated control (20.8%). However,
treated Siha cells at concentrations 40 mM and 80 mM showed cell
cycle distribution Fig. 11(eeg), 69%, 73.1% respectively arrest in G0/
G1 as compared to untreated control (60%). Thus, these data sug-
gested that compound (3) induces the cell cycle arrest G2/M phase
and G0/G1 in HepG2 and Siha cells respectively.
5
ꢁ1
the value of binding constant (K
b
)1 ꢀ 10 l mol . The lower value of
3.3. Cytotoxicity studies
K
b
comparison to classical intercalators may be attributed to non-
planarity in molecules [44] interestingly, same findings (grove
3.3.1. MTT assay
binding) have been observed in the docking studies.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) is a vital dye reduced by the succinate dehydrogenase
3
3
.2. Cell cycle perturbation
enzyme of mitochondrial living cells to produce water insoluble
purple formazan crystals [46,47]. MTT assay has widely been used
to demonstrate the cytotoxic effect of synthetic compounds/Natu-
ral compounds/Extract [48,49]. In the present study, MTT assay
Fig. 12(aee) revealed that compound (3) demonstrated cytotoxic
effects against HepG2, Siha, Hela, MCF-7 and HEK-293 cells at
different doses. The compound (3) displayed potent cytotoxic effect
.2.1. Result & discussion
To assess the effect of compound (3) on cell cycle perturbation,
the flow cytometry assay technique was performed against HepG2
and Siha cell lines. In present study, treated HepG2 cells at con-
centration ranges 10, 20 and 50 mM demonstrated the cell cycle

106
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
Fig. 8. Experimental and theoretical IR of compound(3).
(
IC50 ¼ 7.5
against Hela, Siha and MCF-7 cells were found to be 74.83
6.98 M and 52.0 M Fig. 12(aee). However, compound (3)
showed lesser toxicity against HEK-293 (287.89 M). The IC50 value
m
M) against HepG2 for 48 h treatment and, the IC50
observed as ꢁ7.18 kcal/mol. It was observed that the molecule
mM,
preferred to interact with base pair of DNA and base of chain B and
A as shown in Fig. 13(a). From the docking studies, it was also
observed that molecule made a claw to inhibit the growth of DNA
chain which might be better inhibitor as of cancer cells. Several
polar and no-polar Fig. 13(b) interactions as given Table TS3. The
oxygen atoms of thiazolidinone rings of compound (3) form four
hydrogen bonds with moieties of chains A and B respectively. These
typical docking observations were in good agreement with DNA
binding experimental results. The phenomena can be explained by
considering the non-covalent interactions such as hydrogen bonds,
Van der Waal's forces, electrostatic and hydrophobic bonds. The
docking energy (DGbinding) produced by AutoDock is sum of
various factors as:
6
m
m
m
of compound (3) against cancerous and non-cancerous cell also
depicted in Table 5. Our results indicated that compound (3) was
most active against HepG2 Cells followed by MCF-7, Siha and Hela,
and lesser toxicity against HEK-293. So, compound (3) can be
proven as better anticancer therapeutic agents.
3
3
.4. Molecular simulation
.4.1. DNA docking study
The computational studies have become a very prominent tool
for drug discovery [50]. This is due to its low cost, rapid outcome,
easy and free availability of many software's [51]. In order to verify
our experimental results, DNA docking studies have been carried
out using AutoDock tool [52,53]. The docking study of the com-
pound was performed with DNA dodecamersd(CGCGAATTCGCG)2
D
G
binding
¼
D
G
vdw
þ
D
G
eleþ
D
G
h bondþ
D
G
desolv
þ
DG
tor
Interestingly, it can be seen that, the sum of
Vdw þ Hb þ dissolvation energy is quite high as ¡9.40 kcal/mol.
Electrostatic energy is ¡0.16 kcal/mol. Torsional free energy is
(
PDB ID:1BNA). The binding energy of the docked compound was

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
107
Table 4
Comparison between experimental and theoretical (FT-IR) wave numbers (cmꢁ1) of compound(3) calculated by B3LYP level of theory.
Exp.wave no. in
cm
Calc.(Scaled)
wave no. in
cm
Assignments
ꢁ
1
ꢁ
1
5
5
5
5
58
33
23
14
t
d
y
d
y
d
d
d
t
t
(C34eC33eC35eC35eC36)20þ
(C48eC47eS5)22þ (C24eC20eN7eC28)15þ
(S1eC32) 13þ (S5eC47)13þ (C48eC47eS5)13
(C20eC28eN7) 17þ (C52eC50eN10)17
(S1eC32)28þ (S5eC47)28þ (C33eC32eS1)35
t
(C39eC38eC41eC40)20þ
t
(C44eC43eC40eC41)20þOut(C41eC38eC45eC40)31
s
s
(C56eC52eN10eC50)15
y
d
d
639
674
y
d
648
644
624
604
(C48eN10eC49)11þ
d
(C36eC38eC40)11þ
d(C40eC41eC43)11þ
d (C14eC16-c18)11
(C4eC16eC18)21þ(C14eC16eC18)21þ
(C4eC18eC16)44þ(C11eC14eC16)25þ
d
d
(C61eC63eC65)21þ (C65eC67eC69)21
d
(C4eC18eC16)19þ
d
(C14eC16eC18)25
(C30eN7eC31eC32) 10þ
t
(C47eC48eN10eC49)12
(H62eC61eC63eC65)15þ
6
6
7
65
98
39
709
(H15eC14eC16eC18)15þ
t
t
(H64eC63eC65eC67)15þ
t (H68eC67eC69eC60)15
742
y
(C32eC33)20þ
y
(C33eC35)20þ
y
(C35eC36)20þ
y
(C40eC41)20þ
y (C40eC45)20
735
725
770
s
s
t
s
t
(O2eC32eN7eC31)80
(O2eC32eN7eC31)46þ
(H9eC8eC11eC14)29þ
(O2eC32eN7eC31)35þ
(H9eC8eC11eC14)26þ
(H9eC8eC11eC14)20þ
s
(O6eC47eN10eC48)47
t
t
t
t
(H12eC11eC14eC16)29þ
(C8eC11eC14eC16)þ (C32eC3eC34eC35)19þ
(H15eC14eC16eC18)53
(H12eC11eC14eC16)20þ
t
(H15eC14eC16eC18)29
7
7
69
99
752
774
t
t
(H36eC37eC38eC40)13
785
t
t
(H15eC14eC16eC18)20þ
t(H17eC16eC18eC4)
2
t
0þ
(H36eC37eC38eC40)70
(C50eC52)24þ (C33eC35)13þ
(C36eC38)13
(H36eC37eC38eC40)86
(C20eC28)19þ (C24eC28)19þ
(C60eC61eC63)12þ
t(H62eC61eC63eC65)20
829
834
831
y
y
t
y
d
d
t
t
d
d
d
y
t
t
y
y
(C35eC36)13þ
(C50eC52)19þ
8
8
28
07
y
y
y(C50eC56)19
8
58
04
863
d
(C61eC63eC65)12þ
d
(C63eC65eC67)12þ(
d
C4eC18eC16)12þ
d (C11eC14eC16)12
8
8
59
49
(C4eN3eC30)10þ
d
(C49eN13eC60)10þ
d
(N3eC30eN7)10þ (N10eC49eN13)10
d
(H9eC8eC11eC14)82
(H9eC8eC11eC14)77
9
918
9
8
05
99
(C4eN3eC30)13þ
d
(C28eN7eC31)13þ
d
(C4eN3eC30)13þ
d
(N3eC30eN7)13þ
d (N10eC49eN13)13
(C32eC31eO2)10þ (C47eC48eO6)10þ
(N3eC30eN7)11
911
905
(C33eC35)22þ
(H17eC16eC18eC4)56þ
(H55eC52eC50eC56)29þ
(H56eC58eC50eC52)10
(N10eC50)26
y
(C35eC36)22þ
(H12eC11eC14eC16)14
(H57eC56eC50e C52)29
y
(C36eC38)22þ
y
(C38eC40)22þ
y
(C40eC41)22
968
959
942
t
t
(H57eC56eC50eC52)11þ
t
9
9
48
50
954
947
y
t
t
y
d
d
y
d
d
y
y
d
d
d
y
d
y
d
y
d
d
y
y
y
y
y
d
t
t
t
d
y
d
d
y
y
(C24eC28)43þ
y
(H51eC50eN10eC48)37þ
(H34eC33eC35eC36)74
(H34eC33eC35eC36)78
t (H53eC52eC50eC56)37
9
9
40
36
973
1054
(C11eC14)31þ
d
(C14eC16)31þ
(C14eC16)36
(C14eC16)12þ (C11eC14)12þ
t (C16eC18)31
1052
1048
1029
1170
1159
1136
1105
(H15eC16eC16)12þ
y
(C11eC14)12þ
y
y
y
(C16eC18)12þ
y
y
(C11eC14)12þ
y (C61eC63)12
(C36eC38eC40)74
(H39eC28eC40)40þ
y (H33eC37eC38)40
(C20eC28)49þ
y
(C50eC52)49
(N7eC30)14þ
(H14eC15eC16)34þ (H68eC67eC69)25
(H62eC61eC63)22þ (H64eC63eC65)22þ
(N3eC4)14þ
y(N3eC30)14þ
y
y (N7eC31)14
(H9eC8eC11)34þ
d
d
1186
1192
(H17eC16eC18)13þ
d
y
(H66eC65eC67)22
1185
1266
1255
(H9eC8eC11)71
(C32eC33)42þ
y
(C33eC35)42þ
d
(H36eC37eC38)19
(C33eC35eC36)14þ
y
(C33eC35)25þ (C36eC38)25þ
y
(C45eC47)25
1222
1219
1217
1201
1332
1327
1326
1315
1385
1378
1377
1377
(H42eC41eC43)22þ
d(H34eC33eC35)22þ
y(H37eC36eC38)22
(N3eC4)17þ
t(H58eC56eC50eC52)13þ
d
(H20eC21e22)11þ
(H39eC38e40)13
(H12eC11eC14)29
d (H22eC20eC23)11
(H44eC41eC43)25þ
(H12eC11eC14)31þ
d
(H34eC33eC35)13þ
d
d(H9eC8eC11)29þ
d
(C33eC35)32þ
(C61eC63)27þ
(C60eC61)35þ
(N3eC4)51
y
y
y
(C35eC36)32
(C63eC65)
(C4eC18)35
(C32eC33)11þ
y
(C33eC35)11þ
y
(C40eC41)11þ
y
(C45eC47)11þ
y (C41eC43)12
(H33eC34eC35)77
(H29eC28eN7eC30)36þ
(H26eC24eC28eC20)20þ
(H29eC28eN7eC30)20
(H21eC20eH22)81
t
(H51eC50eN10eC48)11
t(H27eC24eC28eC20)þ
1402
1454
1488
1525
1592
1629
1413
1463
1490
1532
1588
1630
(C36eC38)44þ
y
(C41eC43)44
(C11eC14eC16)þ
(H12eC11eC14)46
(C12eC12eC14)11þ
d
d(C14eC16eC18)11þ
d
(C60eC61eC63)þ
d(C61eC63eC65)11 d (C63eC65eC67)11
(H9eC8eC11)46þ
(C32eC33)65
(C4eC18)56
d
(
continued on next page)

108
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
Table 4 (continued )
Exp.wave no. in
cm
Calc.(Scaled)
wave no. in
cm
Assignments
ꢁ
1
ꢁ
1
1
704
1783
y
y
y
y
y
y
(O2eC31)71
(N4eC4)74,
(N3eC4)73
(C20eH21)86
(C52eH53)83
(C20eH21)76
1
1
720
720
2
3
3
977, 2936
054
089
3055
3055
3059
y-stretching; d-bending; t-torsion, s-out of plane.
shown in Fig. 13(a). The keen evaluation and 3-D visualization of
compound (3) model Fig. 13(a) showed that N-substituted phenyl
ring, 4-thiazolidinone ring and 5-aryl groups are present in the
minor grooves and the remaining parts are outside the grooves and
binds with Chain B of DNA. The oxygen atom forms hydrogen bonds
with Guanine-cytosine and Adenine-thymine base pairs
Table TS3. So, it can be simply concluded that hydrogen bonding
was the major force for the interactions of the compound (3) with
DNA. The Vander Waal's force and steric effects also have a signif-
icant role in binding of ligand to the DNA. From the above results it
could be concluded that the compound (3) has a greater affinity
towards DNA, which is in accordance with the experimental
UVeVisible spectroscopic data [56].
4. Conclusion
Compound (3) adopted (Z, Z)-configuration. The simulated DFT
Fig. 9. Electron density mapped with electrostatic potential surface results to MEP.
studies revealed that the experimental and theoretical in-
vestigations are in good agreement. The ionization energy of the
compound (3)The binding energy of docked molecule with DNA
2
.39 kcal/mol. Finally docking energy is ¡7.18 kcal/mol. It can be
(
1BNA) was found to be ¡7.18 kcal/mol. The DNA binding constant
concluded that DNA binding is in good agreement with docking
studies. The theoretical Free energy, Internal energy and Entropy
was calculated to be ¡1369.23 kcal/mol, ¡5.0 kcal/mol and
5
¡1
was found to be 1.0 x 10 Lmol . MTTassay reveals that compound
3) showed potential inhibitory activity against cancerous cells
(
HepG2 pursued by MCF-7, Siha and Hela cells, and non-toxic effect
against non-cancerous cells HEK-293. Cell cycle analysis (FACS)
technique applied to HepG2 and Siha cells describe that G2/M cell
cycle arrest was found in HepG2 cells, and G0/G1 cell arrest in Siha
cells. Hence, it can be concluded that compound (3) possesses
potent anti-cancer activity against HepG2 cell line.
4
.58 kcal/mol/K at Temperature, T ¼ 298.15 K.
3.4.2. Mechanism of action at supramolecular level
UVeVisible spectroscopic data exhibited that the compound (3)
forms adducts with DNA due to covalent and non-covalent bind-
ings. The docking studies also showed that the lead compound (3)
interacted with the nucleic acid in the minor grooves of DNA. A
deep imminent of interactions at supramolecular level was visu-
alized and developed by docking studies. For this purposes 3-D
docking models were developed for compound (3) and has been
5. Experimental protocol
All the required chemicals were purchased from Merck and
Aldrich Chemical Company (USA). Precoated aluminium sheets
Fig. 10. (aeb): Absorption spectra of compound (3); (a) DNA binding spectra of compound (3), (b) DNA binding spectra of compound in the presence of increasing amount of Ct-
2
ꢁ1
DNA. Inset: plots of [DNA]/(
2
3
aꢁ f
3
) (M cm ) versus [DNA] for the titration of Ct-DNA with compounds. Experimental data points; full lines, linear fitting of the data. (Compound)
.0 ꢀ 10ꢁ M, [DNA] 0.3e1.7 ꢀ 10 M.
4
ꢁ5

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
109
Fig. 11. (aed): Cell cycle phase distribution against HepG2 cells with treatment of compound (3) having concentration 10
distribution against Siha cells at concentrations 40 M & 80 M for 48 h.
mM, 20
m
M and 50
mM for 48 h (eeg): Cell cycle phase
m
m

110
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
Fig. 12. (aee): Percentage cells viability against (a) HepG2 (b) Hela (c) Siha (d) MCF-7 Cells (e) HEK-293.
FT-IR RX1 spectrophotometer as KBr discs. H NMR and 13C NMR
1
Table 5
IC50 Values of the compounds (3) against HepG2, Siha, Hela, MCF-7 and HEK-293
normal human cells lines, and standard against HepG2 and MCF-7 cells.
spectra were recorded on Bruker Spectrospin DPX 300 MHz using
CDCl as a solvent and trimethylsilane (TMS) as an internal stan-
3
S. No
Name of cell lines
Compound [3](IC50
7.5 ( M)
)
Doxorubicin (IC50
)
dard. Splitting patterns are designated as follows; s, singlet; d,
doublet; m, multiplet. Chemical shift values are given in ppm. The
ESI-MS was recorded on micrOTOF-Q II 10330 Electronspray ioni-
zation mass spectrometer (Bruker). X-ray data were collected on
Bruker SMART Apex CCD diffractometer (SAI, Universidade da
Coru n~ a).
1.
2.
3.
4.
5.
HepG2
MCF-7
Hela
Siha
HEK-293
m
3.0(
8.1(
ND
ND
ND
m
m
M)
M)
52.0(
m
M)
M)
M)
M)
66.98(
m
74.83 (
m
287.89(
m
5.1. General procedure for the synthesis of 1-isopropyl
(
Silica gel 60 F254, Merck Germany) were used for thin-layer
phenylthiourea
chromatography (TLC) and spots were visualized under UV light.
Ct-DNA (as sodium salt) was obtained from SRL Pvt. Ltd, Mumbai,
India. The concentrations of DNA were determined spectrometri-
cally with an extinction coefficient of 6600 M cm at 258 nm.
Elemental analysis was carried out on CHNS Elementar analyzer
Phenylisothiocyanate (1 mmol) was dissolved in toluene and
then isopropyl amine (1 mmol) was added slowly at room tem-
perature. After 5 min, the white precipitation appeared. Stirring
was continued for 1 h. The precipitate was collected by filtration,
washed with toluene, and dried to afford product (95.0%) as white
powder.
ꢁ
1
ꢁ1
(
Vario EL-III) and the results were within ±0.3% of the theoretical
values. FT-IR spectra were recorded on Perkin Elmer model 1600

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
111
Fig. 13. (a & b): (a): 3-D representation of polar interactions of compound (3) with DNA(1BNA) (b) and hydrophobic interactions (i&ii) of DNA with compound (3).
5
.1.1. 1-Isoprppyl-3-phenylthiourea (1)
5.3. General procedure of synthesis of bisthiazolidinone derivative
ꢂ
ꢁ1
Yield 95%. m.p: 125e128 C; I.R
lmax (cm ): 3265.14 (eNH),
3
010.03 (CeH, AreH), 1240.83 (C]S); 1HNMR (CDCl
3
)
d
(ppm):
(2.0 mmol)-3-isopropyl-2-(phenylimino)thiazolidin-4-one, was
dissolved in absolute ethanol in a round bottom flask. (1 mmol)
terephthaladehyde was added followed by the addition of
(2.30 mmol) hexahydropyridine to the reaction mixture. The re-
action mixture was refluxed for 11e12 h. The reaction mixture was
cooled to room temperature and the yellow precipitated solid was
collected by filtration, washed with ethanol.The product was
recrystallized in a chloroform and methanol at room temperature.
The shining yellow crystal was obtained.
7
.461 (t, 2H, J ¼ 7.5 Hz, AreH), 7.317 (t, 1H, J ¼ 8.7 Hz, AreH), 7.220
(
d, 2H, J ¼ 7.8 Hz, AreH), 8.267 (s, 1H, NH), 5.873 (d, 1H, J ¼ 6.0 Hz,
13
NH), 4.62e4.55 (m, 1H, CH),1.218 (d, 6H, J ¼ 6.3 Hz, (CH
3
)
2
);
C
3
NMR (CDCl ) d (ppm): 178.94(C]S) (136.07, 130.12, 126.96, 124.88,
3 2
Aromatic), 47.28 (CH), 22.27 ((CH ) C).
5.2. General procedure for the synthesis of thiazolidinone
0
0
0
5
.3.1. (2Z,2 Z,5Z,5 Z)-5,5 -(1,4-phenylenebis(methanylylidene))
bis(3-isopropyl-2-(phenylimino)thiazolidin-4-one) (3)
Yield: 85%; Anal. Calc: C32 ; C 67.82, H 5.34, N 9.89, S
1.32%. found: 67.76, H 5.36, N 9.72, S 11.05%; IR
max (cmꢁ1):
015.20 (AreH), 1692.25 (C]O), 1612.29 (C]N), 744.04 (CeSeC),
(
2.120 g, 16.08 mmol) 1-isopropyl-3-phenylthiourea was dis-
solved in absolute ethanol. (1.643 g, 32.17 mmol) anhydrous so-
dium acetate and (1.190 g, 20.10 mmol) chloroacetic acid were
added in a sequence. The suspension was refluxed for 12 h. The
completion of the reaction was monitored by TLC plate. After the
completion of the reaction, solvent was evaporated on rotator
vacuum. In a reaction mixture, water was added and the aqueous
layer was back-extracted with ethyl acetate. The organic layer was
washed with saturated sodium bicarbonate and brine. The com-
30 4 2 2
H N O S
1
3
1
7
l
1
330.95 (Ter. amine), 1525.70 (C]C); HNMR (CDCl
.631 (s, 2H, HeC]C-) 7.42e7.37 (m, 8H, AreH), 7.25e7.19 (m, 2H,
AreH), 6.99 (d, 4H, J ¼ 7.8 Hz, AreH), 5.07e4.97 (m, 2H, CH), 1.60 (d,
3
) d (ppm);
13
1
1
1
2H, J ¼ 6.3 Hz (CH
49.46 (C]N), 148.26 (C]C), 130.28 (C]CeSe), 134.69, 129.38,
28.70, 124.85, 123.28, 122.56 (aromatic), 48.19 (CeN), 18.97 (CH3);
3 3
)C); C NMR (CDCl ) d (ppm): 166.56 (C]O),
2 4
bined organic extracts were dried over Na SO , filtered through
cotton, and concentrated in vacuum, after the concentration the
mustard-oil like liquid product was obtained which on crystalli-
zation light brown solid was obtained in two weeks.
ESI: m/z: [MþH]þ; 567.186; [Mþ2H]þ: 568.1916, [Mþ3H]þ:
5
69.1845.
6. X-ray crystal structure determination
5
.2.1. 3-Isopropyl-2-(phenylimino)thiazolidin-4-one(2)
Three-dimensional X-ray data were collected on a Bruker Kappa
Apex CCD diffractometer at low temperature for 3 by the f-uscan
ꢂ
ꢁ1
Yield 78%; m.p. 128e130 C; I.R
lmax (cm ): 1715.24 (C]O),
1
1627.65 (C]N), 766.72 (CeSeC), 1370.65 (Ter. amine); HNMR
method. Reflections were measured from a hemisphere of data
ꢂ
(
CDCl
3
)
d
(ppm); 7.36e7.29 (m, 2H, AreH), 7.15e7.08 (m, 1H, AreH),
collected from frames, each of them covering 0.3 in
u
. 108647
6
.952 (d, 2H, J ¼ 7.8 Hz, AreH), 4.91e4.82 (m, 1H, CH), 3.701 (s, 2H,
reflections measured were corrected for Lorentz and polarization
effects and for absorption by multi-scan methods based on
symmetry-equivalent and repeated reflections. Of them, 5630 in-
dependent reflections exceeded the significance level (rFr/
13
CH
71.84 (C]O) 154.08 (eC]Ne), (148.92, 129.12, 124.35, 120.78,
19.73, Aromatic), 47.86 (CH), 24.38 ((CH C).
2
), 1.527 (d, 6H, J ¼ 6.9 Hz, (CH
3 2 3
) C); C NMR (CDCl ) d (ppm):
1
1
3 2
)

112
Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
srFr) > 4.0. After data collection, in each case a multi-scan ab-
Table 6
Crystal Data and Structure Refinement for compound (3).
sorption correction (SADABS) [57] was applied, and the structure
was solved by direct methods and refined by full matrix least-
squares on F2 data using SHELX suite of programs [58] Hydrogen
atoms were located in difference Fourier map and left to refine
freely except for C(7), C(8), C(26) and C(27), which were included in
calculation position and refined in the riding mode. Refinements
were done with allowance for thermal anisotropy of all non-
hydrogen atoms. A final difference Fourier map showed no resid-
Compound code, Identification code
Formula
3
C
32 30 4 2 2
H N O S
Formula weight
T, K
Wavelength, Å
Crystal system
Space group
a/Å
566.72
100
0.71073
Monoclinic
P2 /c
10.9878(7)
9.3077(6)
28.3996(17)
93.170(3)
2900.0(3)
4
1192
1.298
1
ꢁ
3
b/Å
ual density in the crystal: 0.319 and ꢁ0.195 e.Å . A weighting
c/Å
2
2
2
scheme w ¼ 1/[
s
(F
o
) þ (0.044700 P) þ 1.021100 P], was used in
ꢂ
b
/
3
the latter stages of refinement. Further details of the crystal
structure determination are given in Table 6. CCDC 1061909 con-
tains the supplementary crystallographic data for the structure
reported in this paper. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the
Cambridge Crystallographic Data Center, 12 Union Road, Cambridge
CB2 1EZ, UK; fax: (þ44) 1223 336 033; or e-mail: deposit@ccdc.
cam.ac.uk. Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.
V/Å
Z
F
D
m
000
ꢁ3
calc/g cm
ꢁ1
/mm
0.220
1.44 to 26.49
0.0250
ꢂ
q/( )
R
int
3
Crystal size/mm
Goodness-of-fit on F
0.44 ꢀ 0.32 x 0.23
2
1.055
a
R
1
[I > 2
wR (all data)
Largest differences peak and hole (eÅꢁ3)
s(I)]
0.0296
0.0823
0.319 and ꢁ0.195
b
2
2
6
6
016.07.089.
P
P
jj= jF
2
a
R
wR
1
¼
2
jjF
o
j ꢁ jF
c
o
j:
2
P
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
P
.1. Cytotoxicity studies (MTT assay)
b
ꢀ
2
2
2
1=2
¼ f ½wð jF
o
j
ꢁ jF
c
j
Þ ꢄ = ½wðF
ꢁ4
o
Þ ꢄg
:
.1.1. Cell culture
concentrations (0.3e1.7 ꢀ 10 M) of DNA. The intrinsic binding
constant (Kb) was determined by Eq. (2), which was originally
known as BenessieHilderbrand equation and further modified by
Wolfe et al. [62].
Human hepatocellular carcinoma cell line (HepG2), Cervical
cancer cell line (Siha, Hela), Breast cancer cell (MCF-7) and Human
Embryonic Kidney cell line (HEK-293) were procured from National
Curator of Cell Sciences (NCCS) Pune, India. Cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine
[DNA]/(
3
ꢁ
3 ) ¼ [DNA]/(
3
ꢁ
3
) þ 1/K
b
(
3
ꢁ
3
)
(2)
serum with 100 units/mL penicillin, 100
m
g/mL streptomycin, and
ꢂ
2
.5
mg/mL amphotericin B, at 37 C in a relative humidity 80%, 5%
where the apparent absorption coefficients
3
,
3
and
3
correspond
CO2. [59].
to A obs/[compounds], the extinction coefficient for the com-
pounds, and the extinction coefficient for the compounds in the
6.1.2. MTT assay
fully bound form. In plots of [DNA]/(
in the ratio of the slope to intercept.
3
Cytotoxicity of compound (3)/Doxorubicin was evaluated
through MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazo-
lium bromide, M2128 Sigma Aldrich) assay on HepG2, Siha, HeLa,
MCF-7 and HEK-293 cells. MTT is a validated assay for the in vitro
cytotoxicity of any natural, synthetic compounds and extracts
4
[
60,61]. The cell count 1.2 ꢀ 10 cells/well were seeded in 96 well
plate (150
m
L/well). After the overnight incubation, cells were
6.3. DNA docking studies
treated with different concentration of compound (3)/Doxorubicin
for 48 h. After the 48 h of treatment, the medium was remove and
Docking studies were performed at Intel(R) Core(TM) i3 CPU
(2.3 GHz) with XP-based operating system (Windows 2007). 3D
Structure of the lead compound (3) was drawn by Mercury software
using CIF file of crystal structure and saved in pdb file format. The
preparation of the compounds were done by assigning Gastegier
charges, merging non-polar hydrogens, and saving it in PDBQT file
format using AutoDock Tools (ADT)4.2 [54,55]. The X-ray crystal
structure of DNA (PDB ID: 1BNA) was obtained from the Protein Data
Bank [Protein Data Bank. Available online at: http://www.rcsb.org/
pdb]. Using ADT 4.2, DNA was saved in PDB file format leaving
heteroatoms (water). Gastegier charges were assigned to DNA and
saved in PDBQT file format using ADT. Preparation of parameter files
for grid and docking was done using ADT. Docking was performed
with Auto-Dock4.2 (Scripps Research Institute, USA), considering all
the rotatable bonds of ligand as rotatable and receptor as rigid [63]
Grid box size of 47 ꢀ 65 ꢀ 63 Å with 1.0 Å spacing was used that
included the whole DNA. In the present work we describe a plug in
for PyMOL which allows carrying out molecular docking, virtual
screening and binding site analysis with PyMOL. The plug in rep-
resents an interface between PyMOL and two popular docking
programs, Autodock [52,53], Autodock Vina [64] and also the
extensive use of a Python script collection (Autodock Tools) [63].
incubated with 20
buffer) for 4 h. The formazan crystals were formed by mitochon-
drial enzyme reduction, finally solubilized in DMSO (150 L/well)
mL of MTT solution (5 mg/mL in Phosphate saline
m
and absorbance was recorded at 570 nm through the Microplate
reader (iMark, BIORAD, S/N 10321). Percent viability was defined as
the relative absorbance of treated versus untreated control cells.
6.2. DNA binding
The stock solution of disodium salt of Ct-DNA was prepared in
ꢂ
tris-HCl buffer (pH 7.2e7.3) and stored at 4 C temperature. Once
prepared, the stock solution was used within 4 days. The concen-
tration of the solution was determined spectrometrically. The ratio
of absorbance at 260 and 280 (ꢃ1.8) indicated that DNA was suf-
ficiently free of protein. The concentration of DNA was measured
ꢁ
1
ꢁ1
using its extinction coefficient at 260 nm (6600 M cm ) after
dilutions. For the titration purpose, DNA stock solution was diluted
using tris-HCl buffer. The compounds were dissolved in minimum
ꢁ
4
amount of DMSO (2.0 ꢀ 10 M). UV-is absorption spectra were
recorded after each addition of different concentrations of DNA.
Absorption titration was conducted by adding varying

Md. Mushtaque et al. / Journal of Molecular Structure 1127 (2017) 99e113
113
6.4. Methodology of cell cycle analysis
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[
24] A. Rao, J. Balzarini, A. Carbone, A. Chimirri, E.D. Clercq, A.M. Monforte,
Flow cytometry was performed for the analysis of cell cycle ar-
P. Monforte, C. Pannecouque, M. Zappala, Farmaco 5 (2004) 33e39.
rest according to the standard method [65]. The effect of compound
3) on cell cycle on Siha and HepG2 cell lines were analyzed by the
flow cytometry. After reaching the 70% confluency cells were serum
starved overnight and treated with compound (3) for 48 h in
complete medium. The cells were trypsinised, washed two times
with chilled PBS and centrifuged at 1500 rpm for 5 min. Further,
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ml chilled 70% ethanol was added to the cell pellet drop wise and
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vortexed gently. The cells were incubated for 1 h at 4 C. The cells
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[
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l ribonuclease (100 g/mL final concentration) and incubated
for 30 min at 370C. Cells were stained with propidium iodide
50 g/mL final concentrations) and incubated for 15 min. Flow
mL PBS, added
10
m
m
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analyzed by Flowjo software for cell cycle analysis.
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Acknowledgment
One of the authors Md. Mushtaque is highly thankful for
financial assistance from UGC (UGC-BSR fellowship) and Chancellor
Al-Falah University. The Author is also thankful to Dr. Amir Azam,
Deptt. of Chemistry, Jamia Millia Islamia, New Delhi, India for his
help in the process of synthesis and the Head, Deptt. of Biosciences,
JMI, for his permission to work at Genome Biology Lab.
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